1. Field of the Invention
The present invention relates generally to the field of chemical analysis. More particularly, it concerns a new class of nonvolatile, releasable tag reagents for use in the detection and analysis of target molecules i.e., by mass spectrometry.
2. Description of Related Art
Chemical labels, otherwise known as tags or signal groups, are widely used in chemical analysis. Among the types of molecules used are radioactive atoms, fluorescent reagents, luminescent reagents, metal-containing compounds, electron-absorbing substances and light absorbing compounds. Chemical signal groups can be combined with reactivity groups so that they might be covalently attached to the target, the substance being detected. In many cases, however, chemical moieties present on the target may interfere with the detection of the signal group or not allow for measurement of the signal group in an optimal detection environment.
Indirect detection of the target is oftentimes, therefore, preferred. For example, the signal group may be the product of the degradation of the target or a derivative of the target (Bueht et. al., 1974; Senft, 1985; U.S. Pat. Nos. 4,650,750; 4,709,016; 4,629,689). Volatile releasable tag compounds that can be analyzed using various forms of electron-attachment mass spectrometry, often with gas chromatography-mass spectrometry (GC-MS), have been described (Wang et al., 1996; U.S. Pat. Nos. 5,360,819; 5,516,931). Despite the broad range of volatile mass labels reported, a transition from liquid to gas phase is required for analysis which places significant synthetic and size parameters on the label. Isotopic mass labels have also been described, such as using tin or sulfur isotopes, with various mass spectrometric sampling approaches (Arlinghaus et al. 1997; U.S. Pat. No. 5,174,962). The isotopic labeling often limits the extent of multiplexing and provides a more complex analysis requirement.
Mass spectral analysis of signal groups involves none of the concerns related to radioactive signal groups, such as their short half-lives and their safety and disposal issues. Another key advantage to detection of signal groups via mass spectrometry is that it allows a great ability to multiplex, to detect for more than one signal group in a complex mixture, and therefore more than one target at a time. Brummel et al. (1994; 1996) have demonstrated the use of mass spectrometry in the direct analysis of combinatorial libraries of small peptides. However, use of this technology is limited to analysis of the entire reacting compound by mass spectrometry.
Detection of multiple fluorescent labels has been used to analyze nucleic acid sequences. Nucleic acid hybridization probes are modified to contain fluorescent chromophores that when excited by light emit a unique color spectrum signature. In fluorescence based sequencing systems, four different chromophores can be multiplexed within a sample and individually detected with the aid of software deconvolution. The practical upper limit for fluorescence multiplexing is likely to be around 10 different labels due to the broad overlapping spectrum produced by existing fluorescent chromophores. Clearly the development of nonvolatile releasable mass labels, detectable over the usable range of a mass spectrometer, would represent a significant advantage by permitting the multiplexing of tens, hundreds and perhaps even thousands of different mass labels that can be used to uniquely identify each desired target.
At present, while tools are available through which target molecules may be detected, there remains a need for further development of these systems in order to analyze a large number of targets simultaneously. This will allow for the systematic analysis of target molecules with predetermined properties and functions.
It is, therefore, a goal of the present invention to provide compositions and methods relating to the use of release tag compounds for detection and analysis of target molecules.
The present invention relates to the use of nonvolatile, releasable tag compounds, containing releasable mass labels, in chemical analysis, and to the use of these reagents in conjunction with probes which react with or bind noncovalently to a molecule whose presence is to be detected. The releasable tag reagents thus may indirectly detect target molecules, including biomolecular targets. The mass label may be released from the probe following reaction with or binding of the probe to the target and detected by mass spectrometry. The mass value of the label identifies and characterizes the probe and, therefore, the target molecule. In the case of a mass-labeled oligonucleotide probe used to target a polynucleotide, the detection of mass-labels rather than the nucleic acid probes or the nucleic acid targets themselves means that biochemical analysis procedures can be greatly simplified. The need for slow, laborious, costly, and/or complex solid-phase and/or solution-phase cleanup and desalting procedures can be minimized or even eliminated.
Therefore, in accordance with the present invention, there is provided a release tag compound comprising Rx, Re and M, wherein Rx is a reactive group, Re is a release group, and M is a mass label detectable by mass spectrometry. As used herein the term xe2x80x9caxe2x80x9d encompasses embodiments wherein it refers to a single element as well as embodiments including one or more of such elements. For example, the phrase xe2x80x9ca reactive groupxe2x80x9d may refer to a single reactive group, but also encompasses embodiments including more than one reactive group.
Although the mass label may typically be a synthetic polymer or a biopolymer or some combination thereof, in some embodiments, the mass label may generally be any compound that may be detected by mass spectrometry. In particular embodiments, the mass label may be a biopolymer comprising monomer units, wherein each monomer unit is separately and independently selected from the group consisting essentially of an amino acid, a nucleic acid, and a saccharide with amino acids and nucleic acids being preferred monomer units. Because each monomer unit may be separately and independently selected, biopolymer mass labels may be polynucleic acids, peptides, peptide nucleic acids, oligonucleotides, and so on.
As defined herein xe2x80x9cnucleic acidsxe2x80x9d refer to standard or naturally-occurring as well as modified/non-natural nucleic acids, often known as nucleic acid mimics. Thus, the term xe2x80x9cnucleotidesxe2x80x9d refers to both naturally-occurring and modified/nonnaturally-occurring nucleotides, including nucleoside tri, di, and monophosphates as well as monophosphate monomers present within polynucleic acid or oligonucleotide. A nucleotide may also be a ribo; 2xe2x80x2-deoxy; 2xe2x80x2, 3xe2x80x2-deoxy as well as a vast array of other nucleotide mimics that are well-known in the art. Mimics include chain-terminating nucleotides, such as 3xe2x80x2-O-methyl, halogenated base or sugar substitutions; alternative sugar structures including nonsugar, alkyl ring structures; alternative bases including inosine; deaza-modified; chi, and psi, linker-modified; mass label-modified; phosphodiester modifications or replacements including phosphorothioate, methylphosphonate, boranophosphate, amide, ester, ether; and a basic or complete internucleotide replacements, including cleavage linkages such a photocleavable nitrophenyl moieties. These modifications are well known by those of skill in the art and based on fundamental principles as described Saenger (1983), incorporated herein by reference.
Similarly, the term xe2x80x9camino acidxe2x80x9d refers to naturally-occurring amino acid as well as any modified amino acid that may be synthesized or obtained by methods that are well known in the art.
In another embodiment, the mass label may be a synthetic polymer, such as polyethylene glycol, polyvinyl phenol, polyproplene glycol, polymethyl methacrylate, and derivatives thereof. Synthetic polymers may typically contain monomer units selected from the group consisting essentially of ethylene glycol, vinyl phenol, propylene glycol, methyl methacrylate, and derivatives thereof. More typically the mass label may be a polymer containing polyethylene glycol units.
The mass label is typically detectable by a method of mass spectrometry. While it is envisioned that any known mass spectometry method may be used to detect the mass labels of the present invention, methods such as matrix-assisted laser-desorption ionization mass spectrometry, direct laser-desorption ionization mass spectrometry (with no matrix), electrospray ionization mass spectrometry, secondary neutral mass spectrometry, and secondary ion mass spectrometry are preferred.
In certain embodiments the mass label has a molecular weight greater than about 500 Daltons. For some embodiments, it may be preferred to have nonvolatile (including involatile) mass labels; however, for other embodiments volatile mass labels are also contemplated.
As defined herein, the term xe2x80x9creactive groupxe2x80x9d refers to a group capable of reacting with the molecule whose presence is to be detected. For example, the reactive group may be a biomolecule capable of specific molecular recognition. Biomolecules capable of specific molecular recognition may typically be any molecule capable of specific binding interactions with unique molecules or classes of molecules, such as peptides, proteins, polynucleic acids, etc.
Thus, reactive groups disclosed herein for use with the disclosed methods encompass polypeptides and polynucleic acids. As used herein, polypeptides refer to molecules containing more than one amino acid (which include native and non-native amino acid monomers). Thus, polypeptides includes peptides comprising 2 or more amino acids; native proteins; enzymes; gene products; antibodies; protein conjugates; mutant or polymorphic polypeptides; post-translationally modified proteins; genetically engineered gene products including products of chemical synthesis, in vitro translation, cell-based expression systems, including fast evolution systems involving vector shuffling, random or directed mutagenesis, and peptide sequence randomization. In preferred embodiments polypeptides may be oligopeptides, antibodies, enzymes, receptors, regulatory proteins, nucleic acid-binding proteins, hormones, or protein product of a display method, such as a phage display method or a bacterial display method. More preferred polypeptide reactive groups are antibodies and enzymes. As used herein, the phrase xe2x80x9cproduct of a display methodxe2x80x9d refers to any polypeptide resulting from the performance of a display method which are well known in the art. It is contemplated that any display method known in the art may be used to produce the polypeptides for use in conjunction with the present invention.
Similarly, xe2x80x9cpolynucleic acidsxe2x80x9d refer to molecules containing more than one nucleic acid. Polynucleic acids include lengths of 2 or more nucleotide monomers and encompass nucleic acids, oligonucleotides, oligos, polynucleotides, DNA, genomic DNA, mitochondrial DNA (mtDNA), copy DNA (cDNA), bacterial DNA, viral DNA, viral RNA, RNA, message RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), catalytic RNA, clones, plasmids, M13, P1, cosmid, bacteria artificial chromosome (BAC), yeast artificial chromosome (YAC), amplified nucleic acid, amplicon, PCR product and other types of amplified nucleic acid. In preferred embodiments, the polynucleic acid may be an oligonucleotide.
In still further embodiments, Rx is an oligonucleotide having one or more nucleotides, or the oligonucleotide is added after hybridization of Rx to a complementary nucleic acid sequence. The term complementary generally refers to the formation of sufficient hydrogen bonding between two nucleic acids to stabilize a double-stranded nucleotide sequence formed by hybridization of the two nucleic acids.
Typically, nucleotides may be added by a polymerase while oligonucleotides may be added by a ligase. However, it is also contemplated that other methods of adding nucleotides and oligonucleotides known by those of skill in the art may also be employed. In further embodiments, it is provided that the nucleotide added after hybridization may have a chain terminating modification; for example, the added nucleotide may be a chain terminating dideoxy nucleotide.
Embodiments are also provided wherein the added nucleotide or oligonucleotide further comprises a functional group capable of being immobilized on a solid support, for example, a biotin or digoxigenin. Generally, this functional group or binding group or moiety is capable of attaching or binding the tag compound to the solid support. This binding moiety may be attached to the added nucleotide or oligonucleotide directly through an intervening linking group or by specific hybridization to an intermediary oligonucleotide which is itself bound to a solid support. Binding moieties include functional groups for covalent bonding to a solid support, ligands that attach to the solid support via a high-affinity, noncovalent interaction (such as biotin with streptavidin), a series of bases complementary to an intermediary oligonucleotide which is itself attached to the solid support, as well as other means that are well-known to those of skill in the art, such as those described in PCT WO 96/37630, incorporated herein by reference.
In other embodiments, the reactive group may contain a nuclease blocking moiety. These moieties serve to block the digestion of the oligonucleotide by the nuclease, such as an exonuclease. Typical nuclease blocking moieties thus include phosphorothioate, alkylsilyldiester, boranophosphate, methylphosphonate, and peptide nucleic acid.
The mass label is linked, or attached, to the reactive group via a releasable attachment. Thus, typically the mass label is released from all or a part of the reactive group prior to mass spectral analysis as contemplated by the various methods described herein. This releasable attachment typically occurs through the use of a release group which may be the linkage between the mass label and the reactive group or which may comprise a portion or all of the reactive group or which may be contained within the reactive group.
The release group may be any labile group providing for such a releasable attachment. The release group may thus be a chemically cleavable linkage or labile chemical linkage. Such linkages may typically be cleaved by methods that are well known to those of skill in the art, such as by acid, base, oxidation, reduction, heat, light, or metal ion catalyzed, displacement or elimination chemistry. In a particular embodiment, the chemically cleavable linkage comprises a modified base, a modified sugar, a disulfide bond, a chemically cleavable group incorporated into the phosphate backbone, or a chemically cleavable linker. Some examples of these linkages are described in PCT WO 96/37630, incorporated herein by reference. As used herein, xe2x80x9cchemically cleavable linkersxe2x80x9d are moieties cleavable by, for example, acid, base, oxidation, reduction, heat, light, metal ion catalyzed, displacement or elimination chemistry.
Chemically cleavable groups that may be incorporated into the phosphate backbone are well known to those of skill in the art and may include dialkoxysilane, 3xe2x80x2(S)-phosphorothioate, 5xe2x80x2-(S)-phosphorothioate, 3xe2x80x2-(N)-phosphoroamidate, or 5xe2x80x2-(N)phosphoroamidate. In further embodiments the chemically cleavable linkage may be a modified sugar, such as ribose. Alternatively, the linkage may be a disulfide bond.
In still yet another embodiment, Re is contained within Rx. In this case, the release of Re may be activated by a selective event. In particular embodiments, the selective release is mediated by an enzyme such as an exonuclease specific for double-stranded or single-stranded DNA. When it is said that Re is contained within Rx, it will generally be understood that the reactive group contains within its structure the particular release group which will cause the mass label to disconnect from the tag compound in that particular embodiment.
Thus, release groups encompassed by the invention also include groups or linkages cleavable by an enzyme. Enzymatically-cleavable release groups include phosphodiester or amide linkages as well as restriction endonuclease recognition sites.
Preferred embodiments encompass release groups cleavable by nucleases. These nucleases may typically be an exonuclease or a restriction endonuclease. Typical exonucleases include exonucleases specific for both double-stranded and single-stranded polynucleic acids. Additionally, restriction endonucleases encompassed by certain embodiments include Type IIS and Type II restriction endonucleases.
In other embodiments the release group may be cleavable by a protease. Typical proteases include endoproteinases.
Also provided are embodiments wherein Rx comprises a nucleoside triphosphate or is synthesized using mass-labeled nucleoside triphosphates. In another embodiment, Rx comprises a nucleoside phosphoramidite or is synthesized using mass-labeled nucleoside phosphoramidites.
In still further embodiments, mass-labeled probes are provided wherein at least one component is a nucleoside triphosphate. It is further contemplated that the labeled probes of the invention may include at least two unique mass-labels.
Also provided are release tag compounds comprising Rx, Re and M, wherein Rx is a double-stranded oligonucleotide comprising a restriction endonuclease recognition site; Re is a release group comprising a phosphodiester linkage capable of being cleaved by a restriction endonuclease; and M is a mass label detectable by mass spectrometry. Rx may further include a modified nucleotide and the mass label may include a portion of Rx.
Double-stranded oligonucleotides as provided herein include not only two complementary strands hybridized to each other via hydrogen bonding interactions, but also include single strands of nucleotides wherein portions of the strand are single-stranded and portions are double-stranded. For example, portions or all of Rx may include a self-complementary oligonucleotide hairpin where part of Rx is complementary to another part of Rx. In this case, certain conditions allow the formation of a double-stranded duplex between these two portions of Rx. For purposes of certain embodiments of the present invention, it is not necessary that all of Rx need be double-stranded, release tag compounds containing single-stranded regions are also contemplated as being within this embodiment.
Release tag compound are also contemplated having Rx, Re and M, wherein: Rx is a double-stranded oligonucleotide; Re is a chemically cleavable release group; and M is a mass label detectable by mass spectrometry. In this embodiment, Re is typically located within Rx. Cleavage at the chemically cleavable release group is generally inhibited in this aspect by the presence of a double-stranded oligonucleotide at the release group. Previously discussed chemically cleavable release groups, such as 3xe2x80x2-(S)phosphorothioate, 5xe2x80x2-(S)-phosphorothioate, 3xe2x80x2-(N)-phosphoroamidate, 5xe2x80x2-(N)phosphoroamidate, or ribose, may be employed with these embodiments. In these embodiments, a portion of Rx may be rendered single-stranded at Re by hybridization of a portion of Rx to a target nucleic acid.
Also provided is a set of release tag compounds for detecting a particular target nucleic acid. In this aspect, the target nucleic acid typically contains more than one release tag compound. Each release tag compound includes the elements Rx, Re and M, where Rx is an oligonucleotide including a variable region and an invariant region; Re is a release group; and M is a mass label detectable by mass spectrometry. The invariant and variable regions react with the target nucleic acid. It will generally be understood by those of skill in the art that the term xe2x80x9csetxe2x80x9d refers to a group of two or more release tag compounds. Generally each member, i.e., each release tag compound of the group will be different from all other members of the group. That is, each member will include a different combination of reactive group, release group and mass label.
Typically, the mass label of at least one member of the set may identify a specific sequence within the variable region. In some embodiments, the mass label for each member of the set may uniquely identify each different sequence within the variable region. In other embodiments, a combination of the mass labels of two or more release tag compounds may identify each different sequence within the variable region.
As previously discussed, Rx may further comprise a nucleotide or oligonucleotide added after hybridization to the target nucleic acid. In this aspect, the added nucleotide or oligonucleotide may further comprise Rexe2x80x2 and Mxe2x80x2, where Rexe2x80x2 is a release group; and Mxe2x80x2 is a mass label detectable by mass spectrometry. The added nucleotide or oligonucleotide may also contain a chain terminating moiety or a functional group capable of being immobilized on a solid support, such as biotin or digoxigenin.
Methods of producing a mass-labeled probe are provided, comprising combining nucleoside or amino acid monomers with at least one mass-labeled monomer under conditions to allow for polymerization.
Further embodiments are provided wherein the polymerization is mediated by an enzyme. Still further embodiments are provided wherein the polymerization is mediated by chemical synthesis. The preferred synthetic methods to prepare the compound of the present invention are essentially those for standard peptide and DNA synthesis.
For particular embodiments, synthesis in the solid phase is preferred to allow for a wide variety of compounds to be produced using combinatorial methods.
Additional embodiments are provided for a method of producing a mass-labeled probe, comprising the steps of (a) combining nucleoside monomers with at least one activated nucleoside monomer under conditions to allow for polymerization; and (b) adding a releasable, nonvolatile mass unit to said activated nucleoside monomer.
The present invention also provides embodiments which provide a method for detecting a target molecule. Generally, the method includes obtaining a plurality of probes, each probe including a reactive group, a release group and a mass label, as described. It is preferred that each probe within the plurality contains a unique mass-label. By xe2x80x9cunique mass labelxe2x80x9d it is meant that each probe within the plurality will have a different mass label from all other probes in the plurality. A plurality will generally be understood to include two or more probes. Next, the target molecule is contacted with the plurality of probes under conditions suitable to allow for the formation of probe:target molecule complexes. The mass-label is released from the probe and the mass of the mass-label is determined. Typically, the mass is indicative of a specific target molecule. In this way, the target molecule can be identified according to the unique combination of mass-labels.
In another aspect, the invention provides a method for detecting a target molecule where the target molecule is amplified to produce an amplified target molecule. The amplified target molecule is then hybridized with a probe such as those described herein above to produce probe:amplified target molecule complexes. The mass label on the probe:amplified target molecule complexes are then released, and the mass of the mass label determined by mass spectrometry.
The target nucleic acid may be amplified by any method known by one of skill in the art, for example, polymerase chain reaction (xe2x80x9cPCRxe2x80x9d), with PCR being a preferred amplification method. The amplification may include a functional group capable of being immobilized on a solid support, such as biotin or digoxigenin. This functional group may be attached to an oligonucleotide primer incorporated into the amplified molecule during the amplification step or it may be attached to a nucleotide incorporated into the amplified target molecule during the amplification step.
Methods are also provided wherein the amplified target molecule is immobilized onto a solid support and any probe not part of a probe:amplified target molecule complex is removed by washing. It will be understood by those of skill in the art that the nature of the recognition of the target molecule by the reactive group will depend on the identity of the target molecule and the reactive group. For purposes of exemplification and not limitation, this recognition may encompass the formation of a double-stranded duplex by hybridization where the reactive group and target molecule are oligonucleotides. The mass label may be released enzymatically or chemically.
It is contemplated that useful enzymes for this embodiment will include nucleases, such as Type II and IIS restriction endonuclease and exonucleases. The envisioned exonucleases may be specific for double-stranded DNA, such as exonuclease III, T4 endonuclease VII, lambda exonuclease, and DNA polymerase. For these embodiments the release of the mass label may be triggered by the hybridization of the probe to the amplification product. In that embodiment the probe would be single-stranded and capable of hybridizing to the target whose presence was to be detected. The exonuclease may also be specific for single-stranded DNA.
Chemically cleavable linkages may comprise a modified base, a modified sugar, a disulfide bond, a chemically cleavable group incorporated into the phosphate backbone, or a chemically cleavable linker and are typically cleaved by acid, base, oxidation, reduction, heat, light, or metal ion catalyzed, displacement or elimination chemistry.
Embodiments are provided wherein the reactive group further comprises a nucleotide or oligonucleotide added after hybridization to the amplification product, amplified target molecule or amplified nucleic acid molecule. These added nucleotides or oligonucleotides may optionally include a functional group capable of being immobilized on a solid support.
For embodiments employing immobilization onto a solid support, one will typically immobilize the reactive group onto the solid support after addition of the nucleotide or oligonucleotide; then, any probes having unbound reactive groups are removed prior to releasing the mass label of any probe belonging to a probe:amplified target molecule complex or probe:target molecule complex.
In these embodiments, the reactive and release groups may be the same or the release group may be contained within the reactive group. The probe may also comprise at least two unique mass labels.
Multiplexing methods are also provided wherein the target molecule is contacted with a plurality of probes. In these instances, each reactive group of the probe may be associated with a unique mass label or it may be associated with a unique set of mass labels. Thus, a target molecule may be detected by the mass spectral detection of a particular mass label or a particular set of mass labels. Where a set of mass labels is employed, the set of mass labels may be attached to the same probe. Alternatively, each member of the set may be attached to a different probe.
Also provided are methods for detecting mismatches wherein the amplified nucleic acid product comprises a double-stranded molecule containing a mismatch, and an exonuclease-blocking functionality at the 3xe2x80x2 ends of the strands. Typically, this method may further comprise cleavage of at least one strand of the double-stranded molecule at the site of the mismatch; and selective releasing of the mass label. Selective releasing. of the mass label may typically be accomplished by digestion of the cleaved strand by a 3xe2x80x2 to 5xe2x80x2 exonuclease, such as exonuclease III.
As used herein, the term xe2x80x9cselective releasingxe2x80x9d comprises the releasing of a mass label from a probe which belongs to a probe:target molecule complex without releasing a mass label from a probe not belonging to such a complex without having to physically partition the two types of probes. However, some embodiments may include both selective releasing and physical partitioning. The described immobilization and washing techniques exemplify a method of physical partitioning.
The mismatch may be cleaved by an enzyme, such as mutHLS, T4 endonuclease VII, mutY DNA glycosylase, thymine mismatch DNA glycosylase, or endonuclease V. The mismatch may also be cleaved by a chemical, such as OsO4, HONH2, or KMnO4.
The invention further provides a method for detecting a target molecule including the steps of: (a) obtaining a probe including a reactive group, a release group and a nonvolatile mass label; (b) contacting a target molecule with the probe to produce probe:target molecule complexes; (c) selectively releasing the mass label from the probe:target molecule complexes to produce released mass labels; and (d) determining the mass of the released mass labels by mass spectrometry.
Typically, similar chemical and enzymatic release methods may be employed with these embodiments. Selective release of the mass label may also be accomplished by employing cleavage means that are inhibited by the presence of a double-stranded oligonucleotide at the said release group. As used in this context, xe2x80x9cat said release groupxe2x80x9d means that base pairing is maintained on both sides of the release group by at least one nucleotide.
In this embodiment, contacting the probe with the target molecule typically results in the release group being present in a single-stranded region because one strand of the probe interacts with the target molecule, for example, by hybridizing to it.
Another aspect of the invention encompasses a method for multiplexing the detection of a target molecule including: (a) obtaining a plurality of probes, each probe including a reactive group, a release group and a mass label; (b) contacting the target molecule with the plurality of probes to produce probe:target molecule complexes; (c) releasing the mass label from any probe belonging to probe:target molecule complexes to produce released mass labels; and (d) determining the mass of any released mass label by mass spectrometry. In this aspect, each reactive group recognizing a specific target molecule is associated with a unique set of mass labels. It may often be preferred that a plurality of target molecules with the plurality of probes.
The members of the set of mass labels may be attached to the same probe or to different probes. Additionally, the same mass label may be a member of sets identifying more than one reactive group. Thus, in this embodiment the set of mass labels, and not the individual mass label, is unique to a particular reactive group. In this embodiment, probes having a reactive group that identifies a particular target may vary in release group and mass label as well as in other respects.
Immobilization and washing techniques may be employed with this embodiment and it may be preferred in some embodiments to immobilize a plurality of target molecules onto the solid support at spaced locations and to then contact them with the mass-labeled probes. Typical target molecules include a polynucleotide, an antigen, a ligand, a polypeptide, a carbohydrate, and a lipid.
In further embodiments it may be preferred to employ sets of mass labels wherein a mass label member of the set represents a particular moiety or functionality or subset of the target molecule. For example, mass label A could correspond to a reactive group composed of Axe2x80x2X2 . . . XN functionalities where A can be anywhere in the reactive group and only represents Axe2x80x2 and may or may not be structurally related to Axe2x80x2 in any way. Thus, detecting mass label results in the detection of a target molecule that recognizes Axe2x80x2, but does not necessarily identify anything else about the structure or composition of the target molecule.
Thus, methods are provided wherein the unique set of mass labels comprises a mass label that indicates the presence of a specified component within the reactive group. Further embodiments also include methods wherein the mass label indicates the presence of the specified component at a specified location within the reactive group. A reactive group comprising n specified components may be associated with a unique set of mass labels having n members where n may typically be from 1 to 1000. Generally, mass labels are individually attached to the reactive group and are identified intact.
A reactive group comprising n specified components may also be associated with a unique set of mass labels having y members wherein n is less than y!/[x!(yxe2x88x92x)!]; and wherein x comprises the number of mass labels per reactive group.
In some embodiments a plurality of probes may each comprise a known reactive group having a known set of mass labels and the plurality of probes may be prepared by combinatorial synthesis. The plurality of target molecules may also comprise a known chemical structure.
Also provided is a method of monitoring gene expression including (a) obtaining a plurality of probes, each including a reactive group, a release group and a mass label; (b) contacting a plurality of target nucleic acids with the plurality of probes to produce probe:target nucleic acid complexes; (c) selectively releasing the mass label from any probe belonging to a probe:target nucleic acid complexes to produce released mass labels; and (d) determining the mass of any released mass label by mass spectrometry.
Typically, the target nucleic acids may have sequences representative of the genes being expressed in a particular cell culture and are present in concentrations related to their mRNA abundance levels. The target nucleic acids may typically comprise mRNA or first-strand cDNA as well as amplified nucleic acid products.
Such amplified nucleic acid products may be produced using PCR, rtPCR, LCR, Qbeta Replicase, SDA, CPR, TAS, NASBA, or multiple rounds of RNA transcription or some combination thereof. Amplification may be used to selectively amplify a subset of the mRNA pool increasing detection signal for these gene products and reducing background from gene products outside of the amplified subset.
Another embodiment encompasses a method of monitoring gene expression including amplifying a subset of an mRNA pool to produce a plurality of amplified nucleic acid products; contacting a plurality of amplified nucleic acid products with a plurality of probes, each probe including a reactive group, a release group and a mass label to produce probe:amplified nucleic acid product complexes selectively releasing the mass label from any probe belonging to a probe:amplified nucleic acid produce complexes to produce released mass labels determining the mass of any released mass label by mass spectrometry.
For this embodiment, one or more probes or amplified nucleic acid products may be capable of being immobilized onto a solid support.
Another aspect of the invention is a method for detecting a target molecule, including contacting a target molecule with a probe including a reactive group, a release group and a nonvolatile mass label to produce probe:target molecule complexes; releasing the mass label from any probe belonging to a complex to produce released mass labels; selectively desorbing the released mass label from the mass spectral matrix such that the probes not belonging to probe:target molecule complexes do not desorb; and determining the mass of the released mass label by mass spectrometry.
For these embodiments, the mass label should desorb more efficiently from the mass spectral matrix than the probe or the mass-labeled probe. Preferred mass spectral matrices include 2,5-dihydroxybenzoic acid, sinapinic acid, or alpha-cyano-4-hydroxycinammic acid.
A method for detecting a target molecule is also provided. This method includes amplifying one or more target nucleic acids to produce amplified nucleic acid products; incorporating one or more molecules including a reactive group, a release group and a nonvolatile mass label into the amplified nucleic acid product during the amplification process; selectively releasing the mass labels incorporated into the amplified nucleic products to produce released mass labels; and determining the mass of the released mass labels by mass spectrometry.
Incorporated molecules may be oligonucleotide primers and nucleoside triphosphates and the amplified nucleic acid products are produced using PCR, rtPCR, LCR, Qbeta Replicase, SDA, CPR, TAS, NASBA, or multiple rounds of RNA transcription or some combination thereof. One or more second molecules, each including a functional group capable of being immobilized on a solid support, may also be incorporated into the amplified nucleic acid products. The functional group may also be used to bind the amplified nucleic acid products to a solid support, and separate incorporated mass labeled molecules from unincorporated mass labeled molecules. It may also be preferable to separate the amplified nucleic acid products from the unincorporated mass labeled molecules, for example, by binding the amplified nucleic acid products to a solid support or by hybridizing the amplified nucleic acid products to a polynucleotide bound to solid support. In the latter case, the bound polynucleotide may be an oligonucleotide, a polyribonucleotide, a plasmid, an M13, a cosmid, a P1 clone, a BAC or a YAC. A plurality of these polynucleotides may also be immobilized onto the solid support at spaced locations.
Also provided is a method for detecting the presence of a target nucleic acid molecule, said method comprising: obtaining a probe comprising a reactive group, a release group and a mass label; contacting the probe to a target nucleic acid molecule to produce probe:nucleic acid molecule complexes; mass modifying the probe:nucleic acid molecule complexes by attaching a nucleotide or oligonucleotide to the probe to produce mass modified mass labels; releasing the mass modified mass labels; and determining the mass of the mass-modified mass labels by mass spectrometry.
Another embodiment encompasses a method for detecting specific biomolecules in an enzyme-linked affinity assay comprising: obtaining a substrate; contacting a target molecule with an affinity ligand-enzyme conjugate to produce an affinity ligand-enzyme conjugate:target molecule complex; contacting the affinity ligand-enzyme conjugate:target molecule complex with the substrate to produce a mass modified product; and determining the mass of the mass modified product by mass spectrometry.
As used herein, xe2x80x9caffinity ligandsxe2x80x9d are groups, molecules, or moieties having an affinity for, or reacting with a particular target molecule, similar to the reactive groups employed with the mass label probes disclosed above. The affinity ligand may be a biomolecule capable of specific molecular recognition, such as a polypeptide or polynucleic acid. Preferred polypeptides include antibodies, enzymes, receptors, regulatory proteins, nucleic acid-binding proteins, hormones, and protein products of a display method, such as products of a phage display method or a bacterial display method.
The enzymes conjugated to these affinity ligands may be any enzyme that catalyze the conversion of the substrate to a product having a different mass, such as restriction endonucleases and proteases. Thus, the mass of the substrate has been modified in the production of the product by the enzyme. Affinity ligand-enzyme conjugates are molecules where the affinity ligand and enzyme have been attached by the formation of covalent or noncovalent interactions, including hydrogen bonds.
In some embodiments it may be preferable to employ a plurality of restriction endonucleases. In these cases, the various endonucleases may be conjugated to the affinity ligand to form several affinity ligand-enzyme conjugates which are then contacted with the target molecule. Similarly, it may be preferable to employ a plurality of affinity ligand-enzyme conjugates having different affinity ligands, enzymes, or both.
The substrate may be any molecule whose conversion to a mass-modified product is accomplished by the enzyme employed such as a polypeptide. For embodiments employing restriction endonucleases, it may therefore comprise a restriction site.